Chlamydia trachomatis and C. psittaci are important pathogens of humans and animals. The most fundamental chlamydial virulence property is the ability to grow within and destroy host cells by means of an obligate intracellular developmental cycle. Very little is known about how chlamydiae grow and survive within cells, and essentially nothing is known about how chlamydiae regulate gene expression within the unusual environment of a eukaryotic host cell. Not a single chlamydial promotor has been defined by functional studies, nor have repressors, activators and enhancers of gene activity been identified. The signals that trigger intracellular developmental events are also unknown. The goal of the proposed research is to understand how gene expression is regulated by chlamydiae as they progress through the developmental cycle. Towards this end, the expression of selected developmental stage-specific and other key G. trachomatisand C. psittaci genes will be examined in an in vitro transcription system. Genes requiring the major chlamydial sigma factor for expression will be identified by determining the effects of antisigma monoclonal antibodies on transcription and by reconstitution experiments with recombinant chlamydial major sigma factor and E. coli core RNA polymerase. The effects of in vitro-generated mutations on transcription will be studied in the system in order to identify the regulatory DNA elements of selected chlamydial genes, and attempts will be made to identify the proteins that bind to these regions and to clone the genes that encode these proteins. In vitro transcription of the extracellular elementary body form of chlamydiae will also be studied in an attempt to define genes which may be active immediately upon entry of chlamydiae into host cells. An understanding of promotor structure and the mechanism of gene regulation should be invaluable if and when a chlamydial genetic system is developed. These studies should also contribute towards the achievement of the long-term objective of this research: understanding how chlamydiae grow within host cells--a necessary and fundamental step in the process by which chlamydiae cause disease.